Company News About HEPES lysis bufferCAS7365-45-9 is used to extract nucleoplasmic protein
HEPES is 4-hydroxyethylpiperazine-ethanesulfonic acid (N’-a-hydroxythylpiperazine-N’-ethanesulfanic acid), a white crystal powder, hydrogen ion buffer, which can control a constant pH range for a long time. The effective buffer range is pH6.8-8.2. Commonly used to prepare protein extraction lysis buffer, cell culture buffer, etc.
The dissociation constant of HEPES is 7.5. When equimolar mixing of HEPES and its Na-HEPES, the pH of the solution is 7.5. Generally, a fixed molar concentration of HEPES is prepared first, and then the pH is adjusted to the specified value with a strong base (generally commonly used NaOH). Here, NaOH only serves to provide OH. It is also possible to use other strong bases such as KOH. When the pH difference is large, use concentrated NaOH. For fine-tuning, use dilute NaOH. If NaOH solid is added directly, the error is too large, and when NaOH is dissolved Exothermic and changing the temperature may affect the accuracy of the pH electrode.
HEPES lysis buffer preparation:
| Lysis Solution A: | Lysis Solution B: | ||
| HEPES | 10mmol/L，pH7.9 | HEPES | 20mmol/L，pH7.9 |
| KCl | 10mmol/L | NaCl | 420mmol/L |
| MgCl2 | 1.5mmol/L | MgCl2 | 1.5mmol/L |
| DTT | 1mmol/L | DTT | 0.5mmol/L |
| 甘油 | 5% | glycerin | 25% |
| EDTA | 0.2mmol/L | EDTA | 0.2mmol/L |
| NP-40 | 1% | ||
| PMSF (Add before use) | 1mmol/L | PMSF （Add after use） | 0.5mmol/L |
| aprotinin | 3mg/L | aprotinin | 5mg/L |
| leupeptin | 3mg/L | leupeptin | 5mg/L |
| pepstainA | 2mg/L | pepstainA | 3mg/L |
Steps:
1. Collect the cells into an EP tube and centrifuge (4000r/min, 5min, 4 degrees).
2. Wash three times with PBS, centrifuge as above, discard the supernatant.
3. Add 100 μl of buffer A, incubate on ice for 10 min, centrifuge (14000 r/min, 1 min), and discard the supernatant.
4. Resuspend the pellet in 60 microliters of Buffer B, mix well, centrifuge on ice for 30min, centrifuge (14000r/min, 15min, 0 degree), collect the supernatant and discard the pellet.
Among them, the role of lysate A is mainly used to release cytoplasmic proteins and membrane proteins, lysate B is used to release nuclear proteins, NP-40 is both a surfactant and a detergent, its role is both It destroys the cell membrane (mild), and can combine with the released protein to prevent precipitation, so most of the cytoplasmic protein and membrane protein can be removed after the supernatant is removed by centrifugation in the first step. After the nuclear protein is extracted, it can be dialyzed with lysate A for 2h and combined for IP; or diluted with other solutions and replaced with concentrated centrifuge tubes for IP or other experiments.
The main raw materials of Desheng's in vitro diagnostic reagents are: 1. Bis buffer Tris, BICINE, HEPES, CAPS, MOPS, TAPS, EPPS, MOPSO, PIPES, PEP; 2. Chemiluminescent reagents luminol, isoluminol, Acridine ester DMAE-NHS, Acridine ester NSP-DMAE-NHS, Acridine salt NSP-SA, Acridine salt NSP-SA-NHS, Acridine hydrazide NSP-SA-ADH, Acridine ester ME-DMAE-NHS ; 3. New Trinder's reagents TOOS, TOPS, ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, MAOS; 4. Anti-irradiation separation gel for blood collection tube additives, sodium heparin, lithium heparin, EDTA-2K3K, EDTA-2NA, promote Coagulant, coagulation powder, etc. In addition, it also produces Virus Transport Media and carbomer 940/980. Welcome friends to come to buy.
